**2.1.2 Analytical methods**

All monitored parameters were analyzed according to the Standard Methods for the Examination of Water and Wastewater (PN-74/C-04578.03; PN-90/C-04586.04; PN-EN 1189:2000; PN-75/C-04616.01; PN-67/A-86430; PN-EN ISO 6878:2006; PN-EN 13346:2002). The measurement of the pH was done using an Hanna Instruments pehameter model HI 9107. Biogas production from the UASB reactors was recorded by a water displacement meter, while biogas composition was analyzed by an electronic analyzer (LMSxi/G4.18, Gas Data Ltd.). All selected reactors performance parameters were analyzed with Fisher F-tests using Statistica 7.1 software (Statsoft Inc.). Differences were considered statistically significant if the 95% confidence interval of the mean of the parameters did not overlap.

Operation period (d) 45 – 69 70 – 105 106 – 153 154 – 219 OLR (kg COD m-3 d-1) 2.0 4.0 7.0 12.0 HRT (h) 24 24 24 24 Contact surface of the packing medium (m2) 0.00175 0.00175 0.00175 0.00175 Packing volume of steel elements (L) 0.0087 0.0087 0.0087 0.0087 Table 1. Operation regimes for the parallel UASB reactors with and without steel elements Both UASB reactors were fed with UF whey permeate from the manufacture of dairy products in Nowy Dwór Gdański, Poland. The characteristics of the wastewater used in this study is shown in Table 2. It was received from the factory once a week, was stored at -20°C and was thawed before used. Prior to being fed into the reactor, the substrate was diluted with tap water in accordance with the required organic loading rate (OLR) to obtain wastewater COD concentrations in the average range of 4 – 24 g COD L-1. Diluting UF whey permeate was maintained at a temperature 4°C until used. The reactors were not

Parameters Range of values Total COD (g L-1) 52 - 55 Lactose (g L-1) 48 - 53 TP (g L-1) 0.58 - 0.62 Phosphate (g L-1) 0.49 - 0.54 pH (when fresh at 20°C) 4.9 – 5.4 Total iron (mg L-1) 0.26 – 0.35

The seeding inoculum was taken from a laboratory mesophilic reactor treating synthetic dairy wastewater. Each UASB reactor was seeded up to biomass content of 70 g total suspended solids - TSS L-1 at a ratio of 20% (by volume). During startup the reactors were operated at an OLR of 1.0 kg COD m-3 d-1 and at a HRT of 48 h for 44 days. During the reactors operation, biogas production and composition (CH4 and CO2), total COD, total phosphorus – TP, phosphate, soluble iron concentration and pH in the effluent were measured three times a week. After the operation time of 219 days, sludge samples from both UASB reactors were

All monitored parameters were analyzed according to the Standard Methods for the Examination of Water and Wastewater (PN-74/C-04578.03; PN-90/C-04586.04; PN-EN 1189:2000; PN-75/C-04616.01; PN-67/A-86430; PN-EN ISO 6878:2006; PN-EN 13346:2002). The measurement of the pH was done using an Hanna Instruments pehameter model HI 9107. Biogas production from the UASB reactors was recorded by a water displacement meter, while biogas composition was analyzed by an electronic analyzer (LMSxi/G4.18, Gas Data Ltd.). All selected reactors performance parameters were analyzed with Fisher F-tests using Statistica 7.1 software (Statsoft Inc.). Differences were considered statistically significant if the 95% confidence interval of the mean of the parameters did not overlap.

supplemented with trace elements.

**2.1.2 Analytical methods** 

Table 2. Chemical characteristics of wastewater used

collected for the determination of TSS content, TP and total iron contents.

Stage 1 Stage 2 Stage 3 Stage 4

The COD removal efficiency, TP removal efficiency, biogas production and composition were markedly influenced by using steel elements as an additional medium in the UASB reaction chamber.

During Stage 1, both UASB reactors reached the steady-state after 25 days of operation. No statistically significant differences (p>0.05) were observed between UASB reactor with steel elements (RFe) and UASB reactor without steel elements (R0) in term of the average COD removal efficiency and biogas production rate (Fig. 2; 3). Nevertheless, RFe indicated higher (p<0.05) removal efficiency in phosphate (86.2%) and TP (81.2%) than R0 in which the analyzed values were 1.8% and 22.8%, respectively (Fig. 3). CH4 content in biogas produced in RFe was as high as 67.1% which was higher by 11.9% than in R0 (p<0.05). In Stage 2 and 3 both UASB reactors demonstrated a stable work, but statistically significant differences in the values of all the monitoring parameters between R0 and RFe were noticed (p<0.05). The duration of each stage were 36 and 48 days, respectively. The average TP removal efficiency and phosphate removal efficiency in RFe were higher by 77.7% and 83.7%, respectively than in R0 during Stage 2, and 68.1% and 73.9%, respectively during Stage 3 (Fig. 3). During Stage 2 and 3 high COD removal efficiencies (95.6%, 94.8%, respectively) were remained in RFe, in contrast to that of 84.2% in Stage 2 and 80.1% in Stage 3 in R0 (Fig. 3). The average CH4 content in biogas of 78.0% and biogas production of 2.59 m3m-3d-1 in RFe, in contrast to that of 60.8% and 0.92 m3m-3d-1, respectively in R0 (p<0.05), were observed during Stage 2. In Stage 3, biogas production increased by 1.12 m3m-3d-1 in R0 and 1.2 m3m-3d-1 in RFe, but it was still significantly higher in RFe (3.79 m3m-3d-1) than in R0 (2.04 m3m-3d-1), p<0.05 (Fig. 2). Moreover in that stage, the highest methane content in biogas of 79.8% in RFe and 68.1% in R0 were achieved (Fig. 2). During the last stage it was found the highest biogas production rate in RFe of 4.01 m3m-3d-1, while 1.86 m3m-3d-1 in R0 was observed (p<0.05). The average

Fig. 2. Biogas production rate and CH4 content in biogas

Feasibility of Bioenergy Production from

packed with steel elements

TP removal with the duration of the experiment.

Ultrafiltration Whey Permeate Using the UASB Reactors 197

reacted with ferrous iron to probably form insoluble vivianite precipitated in the reaction chamber. It can be confirmed by significant increasing of TSS (by 52.1% in RFe) and the accumulative iron ions and phosphorus content detected in the anaerobic granular sludge in RFe at the end of the experimental period. The TP and total iron percentage in the dry matter was 0.314 and 0.0981, respectively, in RFe and 0.019 and 0.0129, respectively, in R0. This results confirmed the anaerobic microbial corrosion occurred in RFe. Choung & Jeon (2000) and Jeon et al. (2003) obtained similar trends for domestic wastewater treatment under anaerobic conditions. Moreover, the colour of anaerobic sludge granules from RFe was black, while from R0 was grey with white conglomerates (Fig. 4). It indicates that the presence of iron determine the colour of granules. The black colour of granules is due to the formation of large amounts of iron sulphide precipitate (Vlyssides et al., 2009). It was seen that the granule diameter in the sludge bed in RFe was smaller than in R0. It was different from the data reported by Vlyssides et al. (2009), who observed a considerable increase of 40% in the

a) b)

During the experimental period high iron concentrations in the RFe effluent were observed. During Stage 1, the highest content of iron was noticed (20.1 mg L-1) and it was consequently decreased to 19.2, 15.8, 14.2 mg L-1 in Stage 2, 3, 4, respectively. The decrease of the total iron in the effluent from the UASB reactor packed with steel elements can indicate the formation of a protective layer on the steel surface. According to Volkland et al. (2001) under certain conditions the vivianite could act as a corrosion-inhibiting layer. Moreover, biofilm-forming bacteria can protect steel from corrosion. With a dense suspension of microorganisms (> 109 cells mL-1) they can protect the steel surface by forming a corrosion-inhibiting layer in consequence of bacterial adsorption and adhesion (Volkland et al., 2001; Yu et al., 2000). Microbial corrosion and the formation of iron precipitates deteriorate the reactive media of steel elements (Karri et al., 2005). It could explain the gradual decrease in phosphorus and

Biogas production rate and CH4 content in biogas were higher in RFe than in R0 in all stages (except Stage 1 where the differences in biogas production between RFe and R0 were not statistically significant). According to Karri et al. (2005) zero valent iron was an electron donor for methanogenesis. It suggested that microbial corrosion of steel elements supported methanogenesis which contributed to the more CH4 and biogas production in RFe. Iron may

Fig. 4. The photography of granular sludge in UASB reactor a) without steel elements, b)

mean granule diameter resulted in iron accumulation in granules.

CH4 content in biogas decreased to 72.3% in RFe, in contrast to that of 64% in R0, and the differences between R0 and RFe were statistically significant (p<0.05) (Fig. 2). It was found decrease in TP removal efficiency in RFe and R0 to 72.2 and 10.1%(p<0.05), respectively. According to this, the phosphate removal efficiencies decreased, too (Fig. 3). COD removal efficiency was lower than in Stage 3 and achieved 88.8% in RFe and 71.8% in R0, p<0.05 (Fig. 3).

Fig. 3. COD, TP and phosphate removal efficiencies

The study demonstrated that the COD removal efficiency was markedly influenced by using steel elements as an additional medium of the UASB reactor. Iron ions generated from the steel elements must have acted as coagulants and were involved in the removal of suspended organic matter. After the operation time of 219 days, sludge samples from both UASB reactors were collected for the determination of TSS, which was higher by 52.1% in RFe than in R0. Moreover, ferrous ions in wastewater could react to form hydroxides which were the sorption areas for suspended organic matter. Additional sorption areas were made by steel elements surface. Enhancement of COD removal efficiency by zero-valent iron processes were reported by Jeon et al. (2003) and Lai et al. (2007). Vlyssides et al. (2009) showed that the addition of ferrous ions in the form of ferrous chloride solution (2% w/v) induced a stable and excellent COD removal efficiency from synthetic milk wastewater, regardless of the increasing in OLR. When the OLR was as high as 10 g COD L-1 d-1, the COD removal efficiency of 98% was achieved.

Anaerobic steel media corrosion significantly improved TP and phosphate removal from UF whey permeate, but the removal efficiency was affected by the duration of experiment because of deterioration of steel media. The concentration of TP decreased as the phosphate

CH4 content in biogas decreased to 72.3% in RFe, in contrast to that of 64% in R0, and the differences between R0 and RFe were statistically significant (p<0.05) (Fig. 2). It was found decrease in TP removal efficiency in RFe and R0 to 72.2 and 10.1%(p<0.05), respectively. According to this, the phosphate removal efficiencies decreased, too (Fig. 3). COD removal efficiency was lower than in Stage 3 and achieved 88.8% in RFe and 71.8% in R0, p<0.05 (Fig. 3).

Phosphate removal efficiency - RFe Phosphate removal efficiency - R0

TP removal efficiency - RFe TP removal efficiency - R0 COD removal efficiency - RFe COD removal efficiency - R0

2 4 712

OLR (g L-1 d-1)

The study demonstrated that the COD removal efficiency was markedly influenced by using steel elements as an additional medium of the UASB reactor. Iron ions generated from the steel elements must have acted as coagulants and were involved in the removal of suspended organic matter. After the operation time of 219 days, sludge samples from both UASB reactors were collected for the determination of TSS, which was higher by 52.1% in RFe than in R0. Moreover, ferrous ions in wastewater could react to form hydroxides which were the sorption areas for suspended organic matter. Additional sorption areas were made by steel elements surface. Enhancement of COD removal efficiency by zero-valent iron processes were reported by Jeon et al. (2003) and Lai et al. (2007). Vlyssides et al. (2009) showed that the addition of ferrous ions in the form of ferrous chloride solution (2% w/v) induced a stable and excellent COD removal efficiency from synthetic milk wastewater, regardless of the increasing in OLR. When the OLR was as high as 10 g COD L-1 d-1, the

Anaerobic steel media corrosion significantly improved TP and phosphate removal from UF whey permeate, but the removal efficiency was affected by the duration of experiment because of deterioration of steel media. The concentration of TP decreased as the phosphate

Fig. 3. COD, TP and phosphate removal efficiencies

COD removal efficiency of 98% was achieved.

Phosphate/TP removal efficiency (%)

70

75

80

85

COD removal efficiency (%)

90

95

100

reacted with ferrous iron to probably form insoluble vivianite precipitated in the reaction chamber. It can be confirmed by significant increasing of TSS (by 52.1% in RFe) and the accumulative iron ions and phosphorus content detected in the anaerobic granular sludge in RFe at the end of the experimental period. The TP and total iron percentage in the dry matter was 0.314 and 0.0981, respectively, in RFe and 0.019 and 0.0129, respectively, in R0. This results confirmed the anaerobic microbial corrosion occurred in RFe. Choung & Jeon (2000) and Jeon et al. (2003) obtained similar trends for domestic wastewater treatment under anaerobic conditions. Moreover, the colour of anaerobic sludge granules from RFe was black, while from R0 was grey with white conglomerates (Fig. 4). It indicates that the presence of iron determine the colour of granules. The black colour of granules is due to the formation of large amounts of iron sulphide precipitate (Vlyssides et al., 2009). It was seen that the granule diameter in the sludge bed in RFe was smaller than in R0. It was different from the data reported by Vlyssides et al. (2009), who observed a considerable increase of 40% in the mean granule diameter resulted in iron accumulation in granules.

Fig. 4. The photography of granular sludge in UASB reactor a) without steel elements, b) packed with steel elements

During the experimental period high iron concentrations in the RFe effluent were observed. During Stage 1, the highest content of iron was noticed (20.1 mg L-1) and it was consequently decreased to 19.2, 15.8, 14.2 mg L-1 in Stage 2, 3, 4, respectively. The decrease of the total iron in the effluent from the UASB reactor packed with steel elements can indicate the formation of a protective layer on the steel surface. According to Volkland et al. (2001) under certain conditions the vivianite could act as a corrosion-inhibiting layer. Moreover, biofilm-forming bacteria can protect steel from corrosion. With a dense suspension of microorganisms (> 109 cells mL-1) they can protect the steel surface by forming a corrosion-inhibiting layer in consequence of bacterial adsorption and adhesion (Volkland et al., 2001; Yu et al., 2000). Microbial corrosion and the formation of iron precipitates deteriorate the reactive media of steel elements (Karri et al., 2005). It could explain the gradual decrease in phosphorus and TP removal with the duration of the experiment.

Biogas production rate and CH4 content in biogas were higher in RFe than in R0 in all stages (except Stage 1 where the differences in biogas production between RFe and R0 were not statistically significant). According to Karri et al. (2005) zero valent iron was an electron donor for methanogenesis. It suggested that microbial corrosion of steel elements supported methanogenesis which contributed to the more CH4 and biogas production in RFe. Iron may

Feasibility of Bioenergy Production from

et al., 2012; Soccol et al., 2010).

DNA (Liu et al., 2007).

whey permeate by *S. cerevisiae* B4.

whey permeate are operated in Ireland (de Glutz, 2009).

Ultrafiltration Whey Permeate Using the UASB Reactors 199

production (Sarkar et al., 2012). In the USA, bioethanol is mainly used as a 10% petrol additive (E10 is the standard petrol fuel, in 2011 introduced E15). In Brazil, it is offered both as a pure fuel (E100) and is blended with conventional petrol with a content of 20 to 25% (E20, E25). In Europe, with the adoption of the Biofuel Directive 2003/30/EC in 2003, the framework conditions were especially created for European bioethanol production. Today France is a leading producer of bioethanol, then Germany, Spain, Sweden and Dutch are the significant producers in Europe (Gnansounou, 2010). Current large scale production of fuel ethanol is mainly based on sugarcane (Brasil), corn (the USA), sugar beet and wheat (Europe), (Balat & Balat, 2009). The recent rise in the prices of food ethanol biomass has shifted in focus towards a possibility of deriving fuel ethanol from any type of biomass, especially cellulosic biomass (corn or wheat straw, sugarcane bagasse, wood, grass) and food waste biomass (organic waste and wastewater from food processing industries) (Sarkar

According to the literature, cheese whey could be a suitable substrate for bioethanol production (Kourkoutas et al., 2002; Zafar & Owais, 2006). Lewandowska & Kujawski (2007) used a solution of dried UF whey permeate as a substrate for semi-continuous ethanol fermentation. Silveira et al. (2005) fermented the solution of UF whey permeate in batch cultures. Ghaly & El-Taweel (1997) developed a kinetic model for continuous ethanol fermentation from lactose. Moreover, in 2008 there were two industrial scale whey-ethanol plants in the United States which produced 8 million gallons of fuel ethanol per year (Ling, 2008). In New Zealand there were whey-ethanol plants with an annual production of about 5 million gallons of ethanol (Ling, 2008). Industrial-scale plants producing bioethanol form

There are many reports of potential applications of yeast strains in ethanol production from UF whey permeate streams, but most of them focused on *Kluyveromyces sp.* due to its ability to directly ferment lactose (Kourkoutas et al., 2005; Ozmhc & Kargi, 2008; Silveira et al., 2005; ). These yeasts generally suffer from low conversion yields (0.4 kg ethanol kg-1 lactose) and are very sensitive to product (ethanol) inhibition at concentrations as low as 20 g L-1 (de Glutz, 2009). An alternative is to employ indirect fermentation yeasts, such as *Saccharomyces cerevisiae*, which show considerably better ethanol fermentation performance (0.520 kg ethanol kg-1 lactose) and much higher alcohol tolerance (100 - 120 g L-1) (Coté et al., 2004; de Glutz, 2009). The disadvantage of using *S. cerevisiae* is the inability to directly ferment lactose. It can be solved by genetic manipulation of yeasts or facilitate the process with a simultaneous lactose hydrolysis, for example by co-immobilization of yeast cells with the enzyme (Coté et al., 2004; Guimarães et al., 2008). Moreover, higher ethanol production could be achieved by application of different stimulation processes, improving biological activity of yeasts. Many researchers have found that ultrasonic stimulation has the function of promoting the activity of enzyme, cell growth and cell membrane permeability (Chisti, 2003; Liu et al., 2003a; Liu et al., 2007; Schläfer et al., 2000). However, application of ultrasonic irradiation at improper intensity or period has destructive impact on cells by disrupting the cell membranes and deactivating biological molecules such as enzymes or

The objectives of the studies were: (1) to investigate bioethanol production from UF whey permeate in continuous fermentation in UASB reactors by *K. marxianus* 499, (2) to evaluate the effects of low intensity ultrasound (20 kHz, 1 W L-1) for ethanol production from UF

play an important role in granulation phenomena and was found to be a component of essential enzymes that carry out numerous anaerobic reactions (Vlyssides et al., 2009; Yu et al., 2000). The conversion of COD to biogas components and bacterial growth may be limited at iron deficient concentrations. However, the accumulation of iron ions may decrease the specific activity of the bacterial groups, including methanogens (Yu et al., 2000). It was reported that high Fe2+ concentration in the anaerobic sludge granules led to decrease of the specific activity of biomass due to the presence of a large amount of minerals deposited within the granules, a significant decrease in the water content in granules, and the possible toxicity of high-concentration Fe2+ accumulated inside the granules (Yu et al., 2000). During the experiment, biogas production rate was not decreased from Stage 1 to 4, which could indicate that the activity of methanogenic bacteria was not inhibited by anaerobic steel corrosion process. The maximum value for biogas rate was 8.22 L d-1 in RFe and 4.2 L d-1 in R0. Najafpour et al. (2008) achieved the biogas production of 3.6 L d-1 for HRT of 48 h with the methane content of 76% from UF whey permeate. Venetsaneas et al. (2009) achieved about 1 L CH4 d-1 and 68% v/v methane content in biogas in the two-stage process for cheese whey fermentation.
